Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

FIELD OF THE INVENTION

The present invention is in the field of transporter proteins that arerelated to the calcium channel transporter subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins that effect ligand transport andnucleic acid molecules encoding such peptide and protein molecules, allof which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

Transporters

Transporter proteins regulate many different functions of a cell,including cell proliferation, differentiation, and signaling processes,by regulating the flow of molecules such as ions and macromolecules,into and out of cells. Transporters are found in the plasma membranes ofvirtually every cell in eukaryotic organisms. Transporters mediate avariety of cellular functions including regulation of membranepotentials and absorption and secretion of molecules and ion across cellmembranes. When present in intracellular membranes of the Golgiapparatus and endocytic vesicles, transporters, such as chloridechannels, also regulate organelle pH. For a review, see Greger, R.(1988) Annu. Rev. Physiol. 50:111-122.

Transporters are generally classified by structure and the type of modeof action. In addition, transporters are sometimes classified by themolecule type that is transported, for example, sugar transporters,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of molecule (a detailed reviewof channel types can be found at Alexander, S. P. H. and J. A. Peters:Receptor and transporter nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 (1997) andhttp://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

The following general classification scheme is known in the art and isfollowed in the present discoveries.

Channel-Type Transporters.

Transmembrane channel proteins of this class are ubiquitously found inthe membranes of all types of organisms from bacteria to highereukaryotes. Transport systems of this type catalyze facilitateddiffusion (by an energy-independent process) by passage through atransmembrane aqueous pore or channel without evidence for acarrier-mediated mechanism. These channel proteins usually consistlargely of a-helical spanners, although b-strands may also be presentand may even comprise the channel. However, outer membrane porin-typechannel proteins are excluded from this class and are instead includedin class 9.

Carrier-Type Transporters.

Transport systems are included in this class if they utilize acarrier-mediated process to catalyze uniport (a single species istransported by facilitated diffusion), antiport (two or more species aretransported in opposite directions in a tightly coupled process, notcoupled to a direct form of energy other than chemiosmotic energy)and/or symport (two or more species are transported together in the samedirection in a tightly coupled process, not coupled to a direct form ofenergy other than chemiosmotic energy).

Pyrophosphate Bond Hydrolysis-Driven Active Transporters.

Transport systems are included in this class if they hydrolyzepyrophosphate or the terminal pyrophosphate bond in ATP or anothernucleoside triphosphate to drive the active uptake and/or extrusion of asolute or solutes. The transport protein may or may not be transientlyphosphorylated, but the substrate is not phosphorylated.

PEP-Dependent, Phosphoryl Transfer-Driven Group Translocators.

Transport systems of the bacterial phosphoenolpyruvate:sugarphosphotransferase system are included in this class. The product of thereaction, derived from extracellular sugar, is a cytoplasmicsugar-phosphate.

Decarboxylation-Driven Active Transporters.

Transport systems that drive solute (e.g., ion) uptake or extrusion bydecarboxylation of a cytoplasmic substrate are included in this class.

Oxidoreduction-Driven Active Transporters.

Transport systems that drive transport of a solute (e.g., an ion)energized by the flow of electrons from a reduced substrate to anoxidized substrate are included in this class.

Light-Driven Active Transporters.

Transport systems that utilize light energy to drive transport of asolute (e.g., an ion) are included in this class.

Mechanically-Driven Active Transporters.

Transport systems are included in this class if they drive movement of acell or organelle by allowing the flow of ions (or other solutes)through the membrane down their electrochemical gradients.

Outer-Membrane Porins (of b-Structure).

These proteins form transmembrane pores or channels that usually allowthe energy independent passage of solutes across a membrane. Thetransmembrane portions of these proteins consist exclusively ofb-strands that form a b-barrel. These porin-type proteins are found inthe outer membranes of Gram-negative bacteria, mitochondria andeukaryotic plastids.

Methyltransferase-Driven Active Transporters.

A single characterized protein currently falls into this category, theNa+-transporting methyltetrahydromethanopterin:coenzyme Mmethyltransferase.

Non-Ribosome-Synthesized Channel-Forming Peptides or Peptide-likeMolecules.

These molecules, usually chains of L- and D-amino acids as well as othersmall molecular building blocks such as lactate, form oligomerictransmembrane ion channels. Voltage may induce channel formation bypromoting assembly of the transmembrane channel. These peptides areoften made by bacteria and fungi as agents of biological warfare.

Non-Proteinaceous Transport Complexes.

Ion conducting substances in biological membranes that do not consist ofor are not derived from proteins or peptides fall into this category.

Functionally Characterized Transporters for which Sequence Data areLacking.

Transporters of particular physiological significance will be includedin this category even though a family assignment cannot be made.

Putative Transporters in which no Family Member is an EstablishedTransporter.

Putative transport protein families are grouped under this number andwill either be classified elsewhere when the transport function of amember becomes established, or will be eliminated from the TCclassification system if the proposed transport function is disproven.These families include a member or members for which a transportfunction has been suggested, but evidence for such a function is not yetcompelling.

Auxiliary Transport Proteins.

Proteins that in some way facilitate transport across one or morebiological membranes but do not themselves participate directly intransport are included in this class. These proteins always function inconjunction with one or more transport proteins. They may provide afunction connected with energy coupling to transport, play a structuralrole in complex formation or serve a regulatory function.

Transporters of unknown Classification.

Transport protein families of unknown classification are grouped underthis number and will be classified elsewhere when the transport processand energy coupling mechanism are characterized. These families includeat least one member for which a transport function has been established,but either the mode of transport or the energy coupling mechanism is notknown.

Ion Channels

An important type of transporter is the ion channel. Ion channelsregulate many different cell proliferation, differentiation, andsignaling processes by regulating the flow of ions into and out ofcells. Ion channels are found in the plasma membranes of virtually everycell in eukaryotic organisms. Ion channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,ion channels, such as chloride channels, also regulate organelle pH. Fora review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

Ion channels are generally classified by structure and the type of modeof action. For example, extracellular ligand gated channels (ELGs) arecomprised of five polypeptide subunits, with each subunit having 4membrane spanning domains, and are activated by the binding of anextracellular ligand to the channel. In addition, channels are sometimesclassified by the ion type that is transported, for example, chlorinechannels, potassium channels, etc. There may be many classes of channelsfor transporting a single type of ion (a detailed review of channeltypes can be found at Alexander, S. P. H. and J. A. Peters (1997).Receptor and ion channel nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 andhttp://www-biology.ucsd.edu/˜msaier/transport/toc.html.

There are many types of ion channels based on structure. For example,many ion channels fall within one of the following groups: extracellularligand-gated channels (ELG), intracellular ligand-gated channels (ILG),inward rectifying channels (INR), intercellular (gap junction) channels,and voltage gated channels (VIC). There are additionally recognizedother channel families based on ion-type transported, cellular locationand drug sensitivity. Detailed information on each of these, theiractivity, ligand type, ion type, disease association, drugability, andother information pertinent to the present invention, is well known inthe art.

Extracellular ligand-gated channels, ELGs, are generally comprised offive polypeptide subunits, Unwin, N. (1993), Cell 72: 31-41; Unwin, N.(1995), Nature 373: 37-43; Hucho, F., et al., (1996) J. Neurochem. 66:1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem. 239: 539-557;Alexander, S. P. H. and J. A. Peters (1997), Trends Pharmacol. Sci.,Elsevier, pp.4-6; 36-40; 42-44; and Xue, H. (1998) J. Mol. Evol. 47:323-333. Each subunit has 4 membrane spanning regions: this serves as ameans of identifying other members of the ELG family of proteins. ELGbind a ligand and in response modulate the flow of ions. Examples of ELGinclude most members of the neurotransmitter-receptor family ofproteins, e.g., GABAI receptors. Other members of this family of ionchannels include glycine receptors, ryandyne receptors, and ligand gatedcalcium channels.

The Voltage-Gated Ion Channel (VIC) Superfamily

Proteins of the VIC family are ion-selective channel proteins found in awide range of bacteria, archaea and eukaryotes Hille, B. (1992), Chapter9: Structure of channel proteins; Chapter 20: Evolution and diversity.In: Ionic Channels of Excitable Membranes, 2nd Ed., Sinaur Assoc. Inc.,Pubs., Sunderland, Mass.; Sigworth, F. J. (1993), Quart. Rev. Biophys.27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron 15: 489-492;Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci., Elsevier,pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123;Doyle, D. A., et al., (1998) Science 280: 69-77; Terlau, H. and W.Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo- orheterooligomeric structures with several dissimilar subunits (e.g.,a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), butthe channel and the primary receptor is usually associated with the a(or a1) subunit. Functionally characterized members are specific for K⁺,Na⁺ or Ca²⁺. The K⁺ channels usually consist of homotetramericstructures with each a-subunit possessing six transmembrane spanners(TMSs). The a1 and a subunits of the Ca²⁺ and Na⁺ channels,respectively, are about four times as large and possess 4 units, eachwith 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs.These large channel proteins form heterotetra-unit structures equivalentto the homotetrameric structures of most K⁺ channels. All four units ofthe Ca²⁺ and Na⁺ channels are homologous to the single unit in thehomotetrameric K⁺ channels. Ion flux via the eukaryotic channels isgenerally controlled by the transmembrane electrical potential (hencethe designation, voltage-sensitive) although some are controlled byligand or receptor binding.

Several putative K⁺-selective channel proteins of the VIC family havebeen identified in prokaryotes. The structure of one of them, the KcsAK⁺ channel of Streptomyces lividans, has been solved to 3.2 Å Aresolution. The protein possesses four identical subunits, each with twotransmembrane helices, arranged in the shape of an inverted teepee orcone. The cone cradles the “selectivity filter” P domain in its outerend. The narrow selectivity filter is only 12 Å long, whereas theremainder of the channel is wider and lined with hydrophobic residues. Alarge water-filled cavity and helix dipoles stabilize K⁺ in the pore.The selectivity filter has two bound K⁺ ions about 7.5 Å apart from eachother. Ion conduction is proposed to result from a balance ofelectrostatic attractive and repulsive forces.

In eukaryotes, each VIC family channel type has several subtypes basedon pharmacological and electrophysiological data. Thus, there are fivetypes of Ca²⁺ channels (L, N, P, Q and T). There are at least ten typesof K⁺ channels, each responding in different ways to different stimuli:voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive [BK_(Ca),IK_(Ca) and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)]. There areat least six types of Na⁺ channels (I, II, III, μ1, H1 and PN3).Tetrameric channels from both prokaryotic and eukaryotic organisms areknown in which each a-subunit possesses 2 TMSs rather than 6, and thesetwo TMSs are homologous to TMSs 5 and 6 of the six TMS unit found in thevoltage-sensitive channel proteins. KcsA of S. lividans is an example ofsuch a 2 TMS channel protein. These channels may include the K_(Na)(Na⁺-activated) and K_(Vol) (cell volume-sensitive) K⁺ channels, as wellas distantly related channels such as the Tok1 K⁺ channel of yeast, theTWIK-1 inward rectifier K⁺ channel of the mouse and the TREK-1 K⁺channel of the mouse. Because of insufficient sequence similarity withproteins of the VIC family, inward rectifier K⁺ IRK channels(ATP-regulated; G-protein-activated) which possess a P domain and twoflanking TMSs are placed in a distinct family. However, substantialsequence similarity in the P region suggests that they are homologous.The b, g and d subunits of VIC family members, when present, frequentlyplay regulatory roles in channel activation/deactivation.

Experimental evidence indicates that voltage gated Ca²⁺ channels may beimplicated in diseases such as Lambert-Eaton myasthenic syndrome, aparaneoplastic neuromuscular disorder in which an autoimmune responsedirected against a small-cell lung tumor crossreacts with antigens inthe neuromuscular junction. For more information, see Rosenfeld, M. R.,et al., Ann. Neurol. 33: 113-120, 1993, PubMed ID : 8494331; andTaviaux, S., et al., Hum. Genet. 100: 151-154, 1997, PubMed ID: 9254841.

The Epithelial Na⁺ Channel (ENaC) Family

The ENaC family consists of over twenty-four sequenced proteins(Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H.Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G.Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997),Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273:9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger,J. -D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are fromanimals with no recognizable homologues in other eukaryotes or bacteria.The vertebrate ENaC proteins from epithelial cells cluster tightlytogether on the phylogenetic tree: voltage-insensitive ENaC homologuesare also found in the brain. Eleven sequenced C. elegans proteins,including the degenerins, are distantly related to the vertebrateproteins as well as to each other. At least some of these proteins formpart of a mechano-transducing complex for touch sensitivity. Thehomologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the firstpeptide neurotransmitter-gated ionotropic receptor to be sequenced.

Protein members of this family all exhibit the same apparent topology,each with N- and C-termini on the inside of the cell, two amphipathictransmembrane spanning segments, and a large extracellular loop. Theextracellular domains contain numerous highly conserved cysteineresidues. They are proposed to serve a receptor function.

Mammalian ENaC is important for the maintenance of Na⁺ balance and theregulation of blood pressure. Three homologous ENaC subunits, alpha,beta, and gamma, have been shown to assemble to form the highlyNa⁺-selective channel. The stoichiometry of the three subunits isalpha₂, beta1, gamma1 in a heterotetrameric architecture.

The Glutamate-Gated Ion Channel (GIC) Family of NeurotransmitterReceptors

Members of the GIC family are heteropentameric complexes in which eachof the 5 subunits is of 800-1000 amino acyl residues in length(Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol.Sci., Elsevier, pp. 3640). These subunits may span the membrane three orfive times as putative a-helices with the N-termini (theglutamate-binding domains) localized extracellularly and the C-terminilocalized cytoplasmically. They may be distantly related to theligand-gated ion channels, and if so, they may possess substantialb-structure in their transmembrane regions. However, homology betweenthese two families cannot be established on the basis of sequencecomparisons alone. The subunits fall into six subfamilies: a, b, g, d, eand z.

The GIC channels are divided into three types: (1)a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate-and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors.Subunits of the AMPA and kainate classes exhibit 35-40% identity witheach other while subunits of the NMDA receptors exhibit 22-24% identitywith the former subunits. They possess large N-terminal, extracellularglutamate-binding domains that are homologous to the periplasmicglutamine and glutamate receptors of ABC-type uptake permeases ofGram-negative bacteria. All known members of the GIC family are fromanimals. The different channel (receptor) types exhibit distinct ionselectivities and conductance properties. The NMDA-selective largeconductance channels are highly permeable to monovalent cations andCa²⁺. The AMPA- and kainate-selective ion channels are permeableprimarily to monovalent cations with only low permeability to Ca²⁺.

The Chloride Channel (CIC) Family

The CIC family is a large family consisting of dozens of sequencedproteins derived from Gram-negative and Gram-positive bacteria,cyanobacteria, archaea, yeast, plants and animals (Steinmeyer, K., etal., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J. Biol.Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol. 242:595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher, W. E.,et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998), Annu.Rev. Physiol. 60: 689-717). These proteins are essentially ubiquitous,although they are not encoded within genomes of Haemophilus influenzae,Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequenced proteinsvary in size from 395 amino acyl residues (M. jannaschii) to 988residues (man). Several organisms contain multiple ClC familyparalogues. For example, Synechocystis has two paralogues, one of 451residues in length and the other of 899 residues. Arabidopsis thalianahas at least four sequenced paralogues, (775-792 residues), humans alsohave at least five paralogues (820-988 residues), and C. elegans alsohas at least five (810-950 residues). There are nine known members inmammals, and mutations in three of the corresponding genes cause humandiseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiaeonly have one ClC family member each. With the exception of the largerSynechocystis paralogue, all bacterial proteins are small (395-492residues) while all eukaryotic proteins are larger (687-988 residues).These proteins exhibit 10-12 putative transmembrane a-helical spanners(TMSs) and appear to be present in the membrane as homodimers. While onemember of the family, Torpedo ClC-O, has been reported to have twochannels, one per subunit, others are believed to have just one.

All functionally characterized members of the ClC family transportchloride, some in a voltage-regulated process. These channels serve avariety of physiological functions (cell volume regulation; membranepotential stabilization; signal transduction; transepithelial transport,etc.). Different homologues in humans exhibit differing anionselectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductancesequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5channels and others exhibit outward rectifying currents with currentsonly at voltages more positive than +20 mV.

Animal Inward Rectifier K⁺ Channel (IRK-C) Family

IRK channels possess the “minimal channel-forming structure” with only aP domain, characteristic of the channel proteins of the VIC family, andtwo flanking transmembrane spanners (Shuck, M. E., et al., (1994), J.Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995), Am. J.Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron 15:489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78: 227-245;Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171). They mayexist in the membrane as homo- or heterooligomers. They have a greatertendency to let K⁺ flow into the cell than out. Voltage-dependence maybe regulated by external K⁺, by internal Mg²⁺, by internal ATP and/or byG-proteins. The P domains of IRK channels exhibit limited sequencesimilarity to those of the VIC family, but this sequence similarity isinsufficient to establish homology. Inward rectifiers play a role insetting cellular membrane potentials, and the closing of these channelsupon depolarization permits the occurrence of long duration actionpotentials with a plateau phase. Inward rectifiers lack the intrinsicvoltage sensing helices found in VIC family channels. In a few cases,those of Kir1.1a and Kir6.2, for example, direct interaction with amember of the ABC superfamily has been proposed to confer uniquefunctional and regulatory properties to the heteromeric complex,including sensitivity to ATP. The SUR1 sulfonylurea receptor (spQ09428)is the ABC protein that regulates the Kir6.2 channel in response to ATP,and CFTR may regulate Kir1.1a. Mutations in SUR1 are the cause offamilial persistent hyperinsulinemic hypoglycemia in infancy (PHHI), anautosomal recessive disorder characterized by unregulated insulinsecretion in the pancreas.

ATP-Gated Cation Channel (ACC) Family

Members of the ACC family (also called P2X receptors) respond to ATP, afunctional neurotransmitter released by exocytosis from many types ofneurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8: 474-483; Soto,F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr. Biol. 160:91-100). They have been placed into seven groups (P2X₁-P2X₇) based ontheir pharmacological properties. These channels, which function atneuron-neuron and neuron-smooth muscle junctions, may play roles in thecontrol of blood pressure and pain sensation. They may also function inlymphocyte and platelet physiology. They are found only in animals.

The proteins of the ACC family are quite similar in sequence (>35%identity), but they possess 380-1000 amino acyl residues per subunitwith variability in length localized primarily to the C-terminaldomains. They possess two transmembrane spanners, one about 30-50residues from their N-termini, the other near residues 320-340. Theextracellular receptor domains between these two spanners (of about 270residues) are well conserved with numerous conserved glycyl and cysteylresidues. The hydrophilic C-termini vary in length from 25 to 240residues. They resemble the topologically similar epithelial Na⁺ channel(ENaC) proteins in possessing (a) N- and C-termini localizedintracellularly, (b) two putative transmembrane spanners, (c) a largeextracellular loop domain, and (d) many conserved extracellular cysteylresidues. ACC family members are, however, not demonstrably homologouswith them. ACC channels are probably hetero- or homomultimers andtransport small monovalent cations (Me⁺). Some also transport Ca²⁺; afew also transport small metabolites.

The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel(RaR-CaC) Family

Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitiveCa²⁺-release channels function in the release of Ca²⁺ from intracellularstorage sites in animal cells and thereby regulate variousCa²⁺-dependent physiological processes (Hasan, G. et al., (1992)Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem.269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487;Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors andChannels (A. G. Lee, ed.), JAI Press, Denver, Colo.., pp 291-326;Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ryreceptors occur primarily in muscle cell sarcoplasmic reticular (SR)membranes, and IP3 receptors occur primarily in brain cell endoplasmicreticular (ER) membranes where they effect release of Ca²+into thecytoplasm upon activation (opening) of the channel.

The Ry receptors are activated as a result of the activity ofdihydropyridine-sensitive Ca²⁺ channels. The latter are members of thevoltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitivechannels are present in the T-tubular systems of muscle tissues.

Ry receptors are homotetrameric complexes with each subunit exhibiting amolecular size of over 500,000 daltons (about 5,000 amino acylresidues). They possess C-terminal domains with six putativetransmembrane a-helical spanners (TMSs). Putative pore-forming sequencesoccur between the fifth and sixth TMSs as suggested for members of theVIC family. The large N-terminal hydrophilic domains and the smallC-terminal hydrophilic domains are localized to the cytoplasm. Lowresolution 3-dimensional structural data are available. Mammals possessat least three isoforms that probably arose by gene duplication anddivergence before divergence of the mammalian species. Homologues arepresent in humans and Caenorabditis elegans.

IP₃ receptors resemble Ry receptors in many respects. (1) They arehomotetrameric complexes with each subunit exhibiting a molecular sizeof over 300,000 daltons (about 2,700 amino acyl residues). (2) Theypossess C-terminal channel domains that are homologous to those of theRy receptors. (3) The channel domains possess six putative TMSs and aputative channel lining region between TMSa 5 and 6. (4) Both the largeN-terminal domains and the smaller C-terminal tails face the cytoplasm.(5) They possess covalently linked carbohydrate on extracytoplasmicloops of the channel domains. (6) They have three currently recognizedisoforms (types 1, 2, and 3) in mammals which are subject todifferential regulation and have different tissue distributions.

IP₃ receptors possess three domains: N-terminal IP₃-binding domains,central coupling or regulatory domains and C-terminal channel domains.Channels are activated by IP₃ binding, and like the Ry receptors, theactivities of the IP₃ receptor channels are regulated by phosphorylationof the regulatory domains, catalyzed by various protein kinases. Theypredominate in the endoplasmic reticular membranes of various cell typesin the brain but have also been found in the plasma membranes of somenerve cells derived from a variety of tissues.

The channel domains of the Ry and IP₃ receptors comprise a coherentfamily that in spite of apparent structural similarities, do not showappreciable sequence similarity of the proteins of the VIC family. TheRy receptors and the IP₃ receptors cluster separately on the RIR-CaCfamily tree. They both have homologues in Drosophila. Based on thephylogenetic tree for the family, the family probably evolved in thefollowing sequence: (1) A gene duplication event occurred that gave riseto Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved frominvertebrates. (3) The three isoforms of each receptor arose as a resultof two distinct gene duplication events. (4) These isoforms weretransmitted to mammals before divergence of the mammalian species.

The Organellar Chloride Channel (O-ClC) Family

Proteins of the O-ClC family are voltage-sensitive chloride channelsfound in intracellular membranes but not the plasma membranes of animalcells (Landry, D, et al., (1993), J. Biol. Chem. 268: 14948-14955;Valenzuela, S et al., (1997), J. Biol. Chem. 272: 12575-12582;. andDuncan, R. R., et al., (1997), J. Biol. Chem. 272: 23880-23886).

They are found in human nuclear membranes, and the bovine proteintargets to the microsomes, but not the plasma membrane, when expressedin Xenopus laevis oocytes. These proteins are thought to function in theregulation of the membrane potential and in transepithelial ionabsorption and secretion in the kidney. They possess two putativetransmembrane a-helical spanners (TMSs) with cytoplasmic N- andC-termini and a large luminal loop that may be glycosylated. The bovineprotein is 437 amino acyl residues in length and has the two putativeTMSs at positions 223-239 and 367-385. The human nuclear protein is muchsmaller (241 residues). A C. elegans homologue is 260 residues long.

Transporter proteins, particularly members of the calcium channelsubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown transport proteins. Thepresent invention advances the state of the art by providing previouslyunidentified human transport proteins.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human transporter peptides and proteins that arerelated to the calcium channel transporter subfamily, as well as allelicvariants and other mammalian orthologs thereof. These unique peptidesequences, and nucleic acid sequences that encode these peptides, can beused as models for the development of human therapeutic targets, aid inthe identification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate transporteractivity in cells and tissues that express the transporter. Experimentaldata as provided in FIG. 1 indicates expression in humans in the liver,adrenal gland, normal and tumorous nervous tissue, adult amygdala, brainmeningioma tissue, denis-drash, adult and fetal brain, placenta, testisand kidney.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcriptsequence that encodes the transporter protein of the present invention.(SEQ ID NO:1) In addition structure and functional information isprovided, such as ATG start, stop and tissue distribution, whereavailable, that allows one to readily determine specific uses ofinventions based on this molecular sequence. Experimental data asprovided in FIG. 1 indicates expression in humans in the liver, adrenalgland, normal and tumorous nervous tissue, adult amygdala, brainmeningioma tissue, denis-drash, adult and fetal brain, placenta, testisand kidney.

FIG. 2 provides the predicted amino acid sequence of the transporter ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thetransporter protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 47 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a transporter protein or part of a transporter proteinand are related to the calcium channel transporter subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman transporter peptides and proteins that are related to the calciumchannel transporter subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these transporter peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the transporter of the presentinvention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known transporter proteins of the calciumchannel transporter subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. The art has clearly established thecommercial importance of members of this family of proteins and proteinsthat have expression patterns similar to that of the present gene. Someof the more specific features of the peptides of the present invention,and the uses thereof, are described herein, particularly in theBackground of the Invention and in the annotation provided in theFigures, and/or are known within the art for each of the known calciumchannel family or subfamily of transporter proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetransporter family of proteins and are related to the calcium channeltransporter subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIGS. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the transporter peptides of the present invention,transporter peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprising the amino acidsequences of the transporter peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetransporter peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated transporter peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the liver, adrenal gland, normal and tumorous nervous tissue,adult amygdala, brain meningioma tissue, denis-drash, adult and fetalbrain, placenta, testis and kidney. For example, a nucleic acid moleculeencoding the transporter peptide is cloned into an expression vector,the expression vector introduced into a host cell and the proteinexpressed in the host cell. The protein can then be isolated from thecells by an appropriate purification scheme using standard proteinpurification techniques. Many of these techniques are described indetail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the transporter peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The transporter peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a transporter peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the transporter peptide. “Operativelylinked” indicates that the transporter peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the transporter peptide.

In some uses, the fusion protein does not affect the activity of thetransporter peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant transporter peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A transporter peptide-encoding nucleic acid canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the transporter peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the transporter peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of a reference sequence is aligned for comparison purposes. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing. Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences. of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as. well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thetransporter peptides of the present invention as well as being encodedby the same genetic locus as the transporter peptide provided herein. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 10 by ePCR, and confirmed with radiationhybrid mapping.

Allelic variants of a transporter peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the transporter peptide aswell as being encoded by the same genetic locus as the transporterpeptide provided herein. Genetic locus can readily be determined basedon the genomic information provided in FIG. 3, such as the genomicsequence mapped to the reference human. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome10 by ePCR, and confirmed with radiation hybrid mapping. As used herein,two proteins (or a region of the proteins) have significant homologywhen the amino acid sequences are typically at least about 70-80%,80-90%, and more typically at least about 90-95% or more homologous. Asignificantly homologous amino acid sequence, according to the presentinvention, will be encoded by a nucleic acid sequence that willhybridize to a transporter peptide encoding nucleic acid molecule understringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the transporter protein of the present invention. SNPs wereidentified at 47 different nucleotide positions. Changes in the aminoacid sequence caused by these SNPs is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference. Some of these SNPs that arelocated outside the ORF and in introns may affect gene transcription.

Paralogs of a transporter peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the transporter peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a transporter peptide encoding nucleicacid molecule under moderate tostringent conditions as more fully described below.

Orthologs of a transporter peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the transporter peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the transporter peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the transporter peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a transporter peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant transporter peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind ligand, abilityto transport ligand, ability to mediate signaling, etc. Fully functionalvariants typically contain only conservative variation or variation innon-critical residues or in non-critical regions. FIG. 2 provides theresult of protein analysis and can be used to identify criticaldomains/regions. Functional variants can also contain substitution ofsimilar amino acids that result in no change or an insignificant changein function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis oralanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085.(1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as transporter activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the transporterpeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a transporter peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the transporter peptide or could be chosenfor the ability to perform a function, e.g. bind a substrate or act asan immunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe transporter peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in transporter peptidesare described in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the transporter peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code; in which a substituentgroup is included, in which the mature transporter peptide is fused withanother compound, such as a compound to increase the half-life of thetransporter peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature transporter peptide, suchas a leader or secretory sequence or a sequence for purification of themature transporter peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in atransporter-effector protein interaction or transporter-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, transporters isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in the liver, adrenal gland,normal and tumorous nervous tissue, adult amygdala, brain meningiomatissue, denis-drash, adult and fetal brain, placenta, testis and kidney.Specifically, a virtual northern blot shows expression in the liver,adrenal gland, normal and tumorous nervous tissues, adult amygdala,brain meningioma tissue, and denis-drash. In addition, PCR-based tissuescreening panels indicate expression in the adult and fetal brain,placenta, testis, and kidney. A large percentage of pharmaceuticalagents are being developed that modulate the activity of transporterproteins, particularly members of the calcium channel subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in humansin the liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. Such uses can readily be determined. usingthe information provided herein, that known in the art and routineexperimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to transporters that are related tomembers of the calcium channel subfamily. Such assays involve any of theknown transporter functions or activities or properties useful fordiagnosis and treatment of transporter-related conditions that arespecific for the subfamily of transporters that the one of the presentinvention belongs to, particularly in cells and tissues that express thetransporter. Experimental data as provided in FIG. 1 indicates that thetransporter proteins of the present invention are expressed in humans inthe liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. Specifically, a virtual northern blot showsexpression in the liver, adrenal gland, normal and tumorous nervoustissues, adult amygdala, brain meningioma tissue, and denis-drash. Inaddition, PCR-based tissue screening panels indicate expression in theadult and fetal brain, placenta, testis, and kidney. The proteins of thepresent invention are also useful in drug screening assays, incell-based or cell-free systems ((Hodgson, Bio/technology, 1992, Sept10(9);973-80). Cell-based systems can be native, i.e., cells thatnormally express the transporter, as a biopsy or expanded in cellculture. Experimental data as provided in FIG. 1 indicates expression inhumans in the liver, adrenal gland, normal and tumorous nervous tissue,adult amygdala, brain meningioma tissue, denis-drash, adult and fetalbrain, placenta, testis and kidney. In an alternate embodiment,cell-based assays involve recombinant host cells expressing thetransporter protein.

The polypeptides can be used to identify compounds that modulatetransporter activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thetransporter. Both the transporters of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thetransporter. These compounds can be further screened against afunctional transporter to determine the effect of the compound on thetransporter activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thetransporter to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thetransporter protein and a molecule that normally interacts with thetransporter protein, e.g. a substrate or a component of the signalpathway that the transporter protein normally interacts (for example,another transporter). Such assays. typically include the steps ofcombining the transporter protein with a candidate compound underconditions that allow the transporter protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the transporter protein and thetarget, such as any of the associated effects of signal transductionsuch as changes in membrane potential, protein phosphorylation, cAMPturnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for ligand binding. Other candidate compounds include mutanttransporters or appropriate fragments containing mutations that affecttransporter function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) transporter activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate transporter activity. Thus, the transport of aligand, change in cell membrane potential, activation of a protein, achange in the expression of genes that are up- or down-regulated inresponse to the transporter protein dependent signal cascade can beassayed.

Any of the biological or biochemical functions mediated by thetransporter can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the transporter can be assayed.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in the liver,adrenal gland, normal and tumorous nervous tissue, adult amygdala, brainmeningioma tissue, denis-drash, adult and fetal brain, placenta, testisand kidney. Specifically, a virtual northern blot shows expression inthe liver, adrenal gland, normal and tumorous nervous tissues, adultamygdala, brain meningioma tissue, and denis-drash. In addition,PCR-based tissue screening panels indicate expression in the adult andfetal brain, placenta, testis, and kidney.

Binding and/or activating compounds can also be screened by usingchimeric transporter, proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a ligand-binding region can be usedthat interacts with a different ligand then that which is recognized bythe native transporter. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the transporter is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the transporter (e.g. binding partners and/or ligands). Thus, acompound is exposed to a transporter polypeptide under conditions thatallow the compound to bind or to otherwise interact with thepolypeptide. Soluble transporter polypeptide is also added to themixture. If the test compound interacts with the soluble transporterpolypeptide, it decreases the amount of complex formed or activity fromthe transporter target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the transporter. Thus, the soluble polypeptide that competes with thetarget transporter region is designed to contain peptide sequencescorresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the transporter protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix,. separated by SDS-PAGE, and the level oftransporter-binding protein found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques. For example, eitherthe polypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a transporter-bindingprotein and a candidate compound are incubated in the transporterprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with thetransporter protein target molecule, or which are reactive withtransporter protein and compete with the target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the target molecule.

Agents that modulate one of the transporters of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of transporter protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the transporter pathway, by treating cells or tissues thatexpress the transporter. Experimental data as provided in FIG. 1indicates expression in humans in the liver, adrenal gland, normal andtumorous nervous tissue, adult amygdala, brain meningioma tissue,denis-drash, adult and fetal brain, placenta, testis and kidney. Thesemethods of treatment include the steps of administering a modulator oftransporter activity in a pharmaceutical composition to a subject inneed of such treatment, the modulator being identified as describedherein.

In yet another aspect of the invention, the transporter proteins can beused, as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054, Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the transporter and are involved in transporteractivity. Such transporter-binding proteins are also likely to beinvolved in the propagation of signals by the transporter proteins ortransporter targets as, for example, downstream elements of atransporter-mediated signaling pathway. Alternatively, suchtransporter-binding proteins are likely to be transporter inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a transporterprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming atransporter-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the transporter protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a transporter-modulating agent, an antisensetransporter nucleic acid molecule, a transporter-specific antibody, or atransporter-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The transporter proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the liver, adrenal gland, normal andtumorous nervous tissue, adult amygdala, brain meningioma tissue,denis-drash, adult and fetal brain, placenta, testis and kidney. Themethod involves contacting a biological sample with a compound capableof interacting with the transporter protein such that the interactioncan be detected. Such an assay can be provided in a single detectionformat or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered transporter activity incell-based or cell-free assay, alteration in ligand or antibody-bindingpattern, altered isoelectric point, direct amino acid sequencing, andany other of the known assay techniques useful for detecting mutationsin a protein. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985. (19966p, and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the transporter protein in which oneor more of the transporter functions in one population is different fromthose in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other ligand-binding regions thatare more or less active in ligand binding, and transporter activation.Accordingly, ligand dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin the liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. Accordingly, methods for treatment includethe use of the transporter protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe transporter proteins. Antibodies can be prepared from any region ofthe peptide as described herein. However, preferred regions will includethose involved in function/activity and/or transporter/binding partnerinteraction. FIG. 2 can be used to identify particularly importantregions while sequence alignment can be used to identify conserved andunique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkane phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate; rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates that thetransporter proteins of the present invention are expressed in humans inthe liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. Specifically, a virtual northern blot showsexpression in the liver, adrenal gland, normal and tumorous nervoustissues, adult amygdala, brain meningioma tissue, and denis-drash. Inaddition, PCR-based tissue screening panels indicate expression in theadult and fetal brain, placenta, testis, and kidney. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the liver, adrenal gland, normal and tumorousnervous tissue, adult amygdala, brain meningioma tissue, denis-drash,adult and fetal brain, placenta, testis and kidney. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. The diagnostic uses can be applied, notonly in genetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in the liver, adrenalgland, normal and tumorous nervous tissue, adult amygdala, brainmeningioma tissue, denis-drash, adult and fetal brain, placenta, testisand kidney. Thus, where a specific protein has been correlated withexpression in a specific tissue, antibodies that are specific for thisprotein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the transporter peptide to a bindingpartner such as a ligand or protein binding partner. These uses can alsobe applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array; Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a transporter peptide or protein of the present invention(cDNA, transcript and genomic sequence).

Such nucleic acid molecules will consist of, consist essentially of, orcomprise a nucleotide sequence that encodes one of the transporterpeptides of the present invention, an allelic variant thereof, or anortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5 KB, 4 KB,3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified partially or substantially) DNA molecules in solution. IsolatedRNA molecules include in vivo or in vitro RNA transcripts of theisolated DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide'sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprise severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety. of purposes, e.g. control of heterologous, gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein. The isolated nucleic acid molecules can encode the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature peptide (when the mature form has more thanone peptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form; facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the transporter peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combination thereofThe nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the transporter proteinsof the present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant .DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30,40, 50, 100, 250or 500 nucleotides in length. The length of the fragment will be basedon its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets, or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 10 by ePCR, and confirmed with radiation hybrid mapping.

FIG. 3 provides information on SNPs that have been found in the geneencoding the transporter protein of the present invention. SNPs wereidentified at 47 different nucleotide positions. Changes in the aminoacid sequence caused by these SNPs is indicated in FIG. 3 and canreadily be determined using the universal genetic code and the proteinsequence provided in FIG. 2 as a reference. Some of these SNPs that arelocated outside the ORF and in introns may affect gene transcription.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 47 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, themap position was, determined to be on chromosome 10 by ePCR, andconfirmed with radiation hybrid mapping.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates that thetransporter proteins of the present invention are expressed in humans inthe liver, adrenal gland, normal and tumorous nervous tissue, adultamygdala, brain meningioma tissue, denis-drash, adult and fetal brain,placenta, testis and kidney. Specifically, a virtual northern blot showsexpression in the liver, adrenal gland, normal and tumorous nervoustissues, adult amygdala, brain meningioma tissue, and denis-drash. Inaddition, PCR-based tissue screening panels indicate expression in theadult and fetal brain, placenta, testis, and kidney.

Accordingly, the probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. The nucleic acid whose level is determined can be DNAor RNA. Accordingly, probes corresponding to the peptides describedherein can be used to assess expression and/or gene copy number in agiven cell, tissue, or organism. These uses are relevant for diagnosisof disorders involving an increase or decrease in transporter proteinexpression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a transporter protein, such as bymeasuring a level of a transporter-encoding nucleic acid in a sample ofcells from a subject e.g., mRNA or genomic DNA, or determining if atransporter gene has been mutated. Experimental data as provided in FIG.1 indicates that the transporter proteins of the present invention areexpressed in humans in the liver, adrenal gland, normal and tumorousnervous tissue, adult amygdala, brain meningioma tissue, denis-drash,adult and fetal brain, placenta, testis and kidney. Specifically, avirtual northern blot shows expression in the liver, adrenal gland,normal and tumorous nervous tissues, adult amygdala, brain meningioma,tissue, and denis-drash. In addition, PCR-based tissue screening panelsindicate expression in the adult and fetal brain, placenta, testis, andkidney.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate transporter nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid, expression ofthe transporter gene, particularly biological and pathological processesthat are mediated by the transporter in cells and tissues that expressit. Experimental data as provided in FIG. 1 indicates expression inhumans in the liver, adrenal gland, normal and tumorous nervous tissue,adult amygdala, brain meningioma tissue, denis-drash, adult and fetalbrain, placenta, testis and kidney. The method typically includesassaying the ability of the compound to modulate the expression of thetransporter nucleic acid and thus identifying a compound that can beused to treat a disorder characterized by undesired transporter nucleicacid expression. The assays can be performed in cell-based and cell-freesystems. Cell-based assays include cells naturally expressing thetransporter nucleic acid or recombinant cells genetically engineered toexpress specific nucleic acid sequences.

The assay for transporter nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the transporterprotein signal pathway can also be assayed. In this embodiment theregulatory regions of these genes can be operably linked to a reportergene such as luciferase.

Thus, modulators of transporter gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of transportermRNA in the presence of the candidate compound is compared to the levelof expression of transporter mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof nucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate transporter nucleic acid expression incells and tissues that express the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in the liver, adrenal gland,normal and tumorous nervous tissue, adult amygdala, brain meningiomatissue, denis-drash, adult and fetal brain, placenta, testis and kidney.Specifically, a virtual northern blot shows expression in the liver,adrenal gland, normal and tumorous nervous tissues, adult amygdala,brain meningioma tissue, and denis-drash. In addition, PCR-based tissuescreening panels indicate expression in the adult and fetal brain,placenta, testis, and kidney. Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for transporter nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thetransporter nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the liver, adrenal gland, normal and tumorousnervous tissue, adult amygdala, brain meningioma tissue, denis-drash,adult and fetal brain, placenta, testis and kidney.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe transporter gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hagnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in transporter nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in transporter genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the transporter gene and thereby to, determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the transporter geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a transporterprotein.

Individuals carrying mutations in the transporter gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 47different nucleotide. positions. Changes in the amino acid sequencecaused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of these SNPs that are locatedoutside the ORF and in introns may affect gene transcription. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 10 by ePCR, and confirmed with radiationhybrid mapping. Genomic DNA can be analyzed directly or can be amplifiedby using PCR prior to analysis. RNA or cDNA can be used in the. sameway. In some uses, detection, of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995,)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a transporter gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutanttransporter gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers, et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-2,95 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144,(1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Meyers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and. selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the transporter gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment. FIG. 3 provides information on SNPs that have been foundin the gene encoding the transporter protein of the present invention.SNPs were identified at 47 different nucleotide positions. Changes inthe amino acid sequence caused by these SNPs is indicated in FIG. 3 andcan readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a reference. Some of these SNPsthat are located outside the ORF and in introns may affect genetranscription.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol transporter gene expression in cells, tissues, and organisms. ADNA antisense nucleic acid molecule is, designed to be complementary toa region of the gene involved in transcription, preventing transcriptionand hence production of transporter protein. An antisense RNA or DNAnucleic acid molecule would hybridize to the mRNA and thus blocktranslation of mRNA into transporter protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of transporter nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired transporter nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the transporterprotein, such as ligand binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in transporter geneexpression. Thus, recombinant cells, which include the patients cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredtransporter protein to treat the individual.

The invention also encompasses kits for detecting the presence of atransporter nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in the liver, adrenal gland,normal and tumorous nervous tissue, adult amygdala, brain meningiomatissue, denis-drash, adult and fetal brain, placenta, testis and kidney.Specifically, a virtual northern blot shows expression in the liver,adrenal gland, normal and tumorous nervous tissues, adult amygdala,brain meningioma, tissue, and denis-drash. In addition, PCR-based tissuescreening panels indicate expression in the adult and fetal brain,placenta, testis, and kidney. For example, the kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting transporter nucleic acid in a biological sample; means fordetermining the amount of transporter nucleic acid in the sample; andmeans for comparing the amount of transporter nucleic acid in the samplewith a standard; The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect transporter protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11 995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of, the transporter proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thetransporter gene of the present invention. FIG. 3 provides informationon SNPs that have been found in the gene encoding the transporterprotein of the present invention. SNPs were identified at 47 differentnucleotide positions. Changes in the amino acid sequence caused by theseSNPs is indicated in FIG. 3 and can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as areference. Some of these SNPs that are located outside the ORF and inintrons may affect gene transcription.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Inmunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified transporter gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in procaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats..

In addition to control regions that promote transcription, expressionvectors may also, include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et .al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterotransporter. Typical fusion expression vectorsinclude pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Mass. ) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Examples ofsuitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology. Methods in Enzymology 185, AcademicPress; San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec 1(Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series.(Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 70:31-39,(1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pNM2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)). The expression vectors listed herein are providedby way of example only of the well-known vectors available to those ofordinary skill in the art that would be useful to express the nucleicacid molecules. The person of ordinary skill in the art would be awareof other vectors suitable, for maintenance propagation or expression ofthe nucleic acid molecules described herein. These are found for examplein Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory, Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein, Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined, tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers, include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotichost cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such astransporters, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with transporters, the protein can be isolated from the hostcell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing atransporter protein or peptide that can be further purified to producedesired amounts of transporter protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe transporter protein or transporter protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native transporter protein is usefulfor assaying compounds that stimulate or inhibit transporter proteinfunction.

Host cells are also useful for identifying transporter protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutanttransporter protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native transporterprotein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA that is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a transporterprotein and identifying and evaluating modulators of transporter proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the transporter proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation. signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the transporter protein to particularcells.

Methods for generating transgenic animals via, embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell; from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the- quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,transporter protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivotransporter protein function, including ligand interaction, the effectof specific mutant transporter proteins on transporter protein functionand ligand interaction, and the effect of chimeric transporter proteins.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or moretransporter protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

1. An isolated peptide consisting of an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence shown in SEQ IDNO:2; (b) an amino acid sequence of an allelic variant of an amino acidsequence shown in SEQ ID NO:2, wherein said allelic variant is encodedby a nucleic acid molecule that hybridizes under stringent conditions tothe opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) an amino acid sequence of an ortholog of an amino acid sequenceshown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the oppositestrand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) afragment of an amino acid sequence shown in SEQ ID NO:2, wherein saidfragment comprises at least 10 contiguous amino acids.
 2. An isolatedpeptide comprising an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence shown in SEQ ID NO:2; (b) anamino acid sequence of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said allelic variant is encoded by anucleic acid molecule that hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) an amino acid sequence of an ortholog of an amino acid sequenceshown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acidmolecule that hybridizes under stringent conditions to the oppositestrand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3; and (d) afragment of an amino acid sequence shown in SEQ ID NO:2, wherein saidfragment comprises at least 10 contiguous amino acids.
 3. An isolatedantibody that selectively binds to a peptide of claim
 2. 4. An isolatednucleic acid molecule consisting of a nucleotide sequence selected fromthe group consisting of: (a) a nucleotide sequence that encodes an aminoacid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence thatencodes of an allelic variant of an amino acid sequence shown in SEQ IDNO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes an ortholog ofan amino acid sequence shown in. SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e), a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO:2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 5. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human transporter protein, saidmethod comprising administering to a patient a pharmaceuticallyeffective amount of an agent identified by the method of claim
 16. 19. Amethod for identifying a modulator of the expression of a peptide ofclaim 2, said method comprising contacting a cell expressing saidpeptide with an agent, and determining if said agent has modulated theexpression of said peptide.
 20. An isolated human transporter peptidehaving an amino acid sequence that shares at least 70% homology with anamino acid sequence shown in SEQ ID NO:2.
 21. A peptide according toclaim 20 that shares at least 90 percent homology with an amino acidsequence shown in SEQ ID NO:2.
 22. An isolated nucleic acid moleculeencoding a human transporter peptide, said nucleic acid molecule sharingat least 80percent homology with a nucleic acid molecule shown in SEQ IDNOS:1 or
 3. 23. A nucleic acid molecule according to claim 22 thatshares at least 90 percent homology with a nucleic acid molecule shownin SEQ ID NOS:1 or 3.